title: Mutations outside the rifampicin resistance-determining region associated with rifampicin resistance in Mycobacterium tuberculosis.
abstract: OBJECTIVE
  Ninety-six percent of rifampicin resistance in Mycobacterium tuberculosis was shown to be associated with mutations inside the 81 bp rifampicin resistance-determining region (RRDR) located in the centre of the rpoB gene. The detection of rifampicin resistance by targeting the RRDR failed to match with a resistant phenotype in 4% of all cases. Our study aims to identify the mutations outside the RRDR that are associated with rifampicin resistance in M. tuberculosis.
  
  
  RESULTS
  Among 50 rifampicin-resistant and 20 rifampicin-susceptible clinical isolates of M. tuberculosis, 2 of the rifampicin-resistant isolates did not harbour any known mutations in the RRDR. Sequencing analysis of the whole rpoB gene identified two rare mutations, V146F and I572F. A molecular structure model based on Thermus thermophilus RpoB revealed that both these substituted amino acids are located in close proximity to the rifampicin-binding pocket of the β-subunit. Substitutions of simple amino acids for bulky ones are likely to affect the protein-drug interaction. Cloning and transformation of the mutated rpoB gene into wild-type Mycobacterium smegmatis and M. tuberculosis successfully elevated the MIC of rifampicin and conferred the rifampicin resistance phenotype.
  
  
  CONCLUSIONS
  Our study showed that amino acid positions 146 and 572 are associated with rifampicin resistance in M. tuberculosis in addition to the RRDR. Molecular assays for identifying rifampicin-resistant M. tuberculosis might be improved in terms of accuracy by including these two positions.
authors.affiliation: Department of Microbiology, The University of Hong Kong, University Pathology Building, Queen Mary Hospital, Hong Kong Special Administrative Region, China.
authors.forename: Gilman Kit Hang
authors.initials: GK
authors.lastname: Siu
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.forename: Terrence C K
authors.initials: TC
authors.lastname: Lau
authors.forename: Ricky W T
authors.initials: RW
authors.lastname: Lau
authors.forename: Pak-Leung
authors.initials: PL
authors.lastname: Ho
authors.forename: Wing-Wai
authors.initials: WW
authors.lastname: Yew
authors.forename: Stephen K W
authors.initials: SK
authors.lastname: Tsui
authors.forename: Vincent C C
authors.initials: VC
authors.lastname: Cheng
authors.forename: Kwok-Yung
authors.initials: KY
authors.lastname: Yuen
authors.forename: Wing-Cheong
authors.initials: WC
authors.lastname: Yam
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000995:Antitubercular Agents / Q000494:pharmacology*; D001665:Binding Sites; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics*; D024881:Drug Resistance, Bacterial; D006801:Humans; D020125:Mutation, Missense; D020102:Mycobacterium smegmatis / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification; D012293:Rifampin / Q000494:pharmacology*; D016963:Thermus thermophilus / Q000235:genetics; D014376:Tuberculosis / Q000382:microbiology
chemical_list: D000995:Antitubercular Agents; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit; D012293:Rifampin